Helena Lucena-Padrós

Genome Sequence of Lactobacillus pentosus IG1, a Strain Isolated from Spanish-Style Green Olive Fermentations

Lactobacillus pentosus is the most prevalent lactic acid bacterium in Spanish-style green olive fermentations. Here we present the draft genome sequence of L. pentosus IG1, a bacteriocin-producing strain with biotechnological and probiotic properties isolated from this food fermentations.

Propionibacterium olivae sp. nov. and Propionibacterium damnosum sp. nov., isolated from spoiled packaged Spanish-style green olives.

Five strains of Gram-stain-positive bacteria were isolated from anomalous fermentations occurring in post-packaging of sealed airtight food-grade plastic pouches of Spanish-style green olives. These isolates could be grouped into two sets, which showed a similarity in their respective 16S rRNA gene sequences of 98.40 and 98.44 % with Propionibacterium acidipropionici NCFB 563 and 98.33 and 98.11 % with Propionibacterium microaerophilum M5(T), respectively, and a similarity of 99.41 % between them. The 16S rRNA gene phylogeny revealed that the isolates grouped into two statistically well-supported clusters separate from P. acidipropionici NCFB 563 and P. microaerophilum M5(T). Enzymic activity profiles as well as fermentation patterns differentiated these two novel bacteria from other members of the genus Propionibacterium. Finally, phenotypic, genotypic and phylogenetic data, supported the proposal of two novel species of the genus Propionibacterium, for which the names Propionibacterium olivae sp. nov. (type strain, IGBL1(T) = CECT 8061(T) = DSM 25436(T)) and Propionibacterium damnosum sp. nov. (type strain, IGBL13(T) = CECT 8062(T) = DSM 25450(T)) are proposed.

Enterococcus olivae sp. nov., isolated from Spanish-style green-olive fermentations.

Six strains of a hitherto unknown, Gram-stain-positive coccus were recovered from samples of Spanish-style green-olive fermentations. The 16S rRNA gene sequences from these isolates shared 98.7% and 98.5% of their nucleotide positions with those from Enterococcus saccharolyticus subsp. taiwanensis 812(T) and from E. saccharolyticus subsp. saccharolyticus ATCC 43076(T), respectively. The sequence of the rpoA gene in the isolates was 95% similar to that of E. saccharolyticus CECT 4309(T) ( = ATCC 43076(T)). The 16S rRNA and rpoA gene phylogenies revealed that the isolates grouped in a statistically well-supported cluster separate from E. saccharolyticus. Enzyme activity profiles as well as fermentation patterns differentiated the novel bacteria from other members of the Enterococcus genus. Finally, phenotypic, genotypic and phylogenetic data supported the identification of a novel species of the genus Enterococcus, for which the name Enterococcus olivae sp. nov. is proposed. The type strain is IGG16.11(T) ( = CECT 8063(T) = DSM 25431(T)).

Diversity and enumeration of halophilic and alkaliphilic bacteria in Spanish-style green table-olive fermentations.

The presence and enumeration of halophilic and alkaliphilic bacteria in Spanish-style table-olive fermentations was studied. Twenty 10-tonne fermenters at two large manufacturing companies in Spain, previously studied through both culture dependent and independent (PCR-DGGE) methodologies, were selected. Virtually all this microbiota was isolated during the initial fermentation stage. A total of 203 isolates were obtained and identified based on 16S rRNA gene sequences. They belonged to 13 bacterial species, included in 11 genera. It was noticeable the abundance of halophilic and alkaliphilic lactic acid bacteria (HALAB). These HALAB belonged to the three genera of this group: Alkalibacterium, Marinilactibacillus and Halolactibacillus. Ten bacterial species were isolated for the first time from table olive fermentations, including the genera Amphibacillus, Natronobacillus, Catenococcus and Streptohalobacillus. The isolates were genotyped through RAPD and clustered in a dendrogram where 65 distinct strains were identified. Biodiversity indexes found statistically significant differences between both patios regarding genotype richness, diversity and dominance. However, Jaccard similarity index suggested that the halophilic/alkaliphilic microbiota in both patios was more similar than the overall microbiota at the initial fermentation stage. Thus, up to 7 genotypes of 6 different species were shared, suggesting adaptation of some strains to this fermentation stage. Morisita-Horn similarity index indicated a high level of codominance of the same species in both patios. Halophilic and alkaliphilic bacteria, especially HALAB, appeared to be part of the characteristic microbiota at the initial stage of this table-olive fermentation, and they could contribute to the conditioning of the fermenting brines in readiness for growth of common lactic acid bacteria.

Genetic diversity and dynamics of bacterial and yeast strains associated to Spanish-style green table-olive fermentations in large manufacturing companies

We have genotyped a total of 1045 microbial isolates obtained along the fermentation time of Spanish-style green table olives from the fermentation yards (patios) of two large manufacturing companies in the Province of Sevilla, south of Spain. Genotyping was carried out using RAPD-PCR fingerprinting. In general, isolates clustered well into the relevant phylogenetic dendrograms, forming separate groups in accordance to their species adscription. We could identify which bacterial and yeast genotypes (strains) persisted throughout the fermentation at each patio. Also, which of them were more adapted to any of the three stages, i.e. initial, middle and final, described for this food fermentation. A number of genotypes were found to be shared by both patios. Fifty seven of these belonged to five different bacterial species, i.e. Lactobacillus pentosus, Lactobacillus paracollinoides/collinoides, Lactobacillus rapi, Pediococcus ethanolidurans and Staphylococcus sp., although most of them (51) belonged to L. pentosus. Four yeast genotypes were also shared, belonging to the species Candida thaimueangensis, Saccharomyces cerevisiae and Hanseniaspora sp. Two genotypes of L. pentosus were found to be grouped with those of two strains used in commercially available starter cultures, one of them bacteriocinogenic, which were used up to three years before this study in these patios, demonstrating the persistence of selected strains in this environment. Biodiversity was assessed though different indexes, including richness, diversity and dominance. A statistically significant decrease in biodiversity between the initial and final stages of the fermentation was found in both patios. However, values of biodiversity indexes in the fermenters were very similar, and no significant differences were found in the total biodiversity between both patios. This study allowed us to identify a range of well adapted strains (genotypes), especially those belonging to the lactic acid bacteria, which could be useful to improve safety and quality of table olive fermentations.

Vibrio olivae sp. nov., isolated from spanish-style green-olive fermentations

Three isolates originating from Spanish-style green-olive fermentations in a manufacturing company in the province of Seville, Spain, were taxonomically characterized by a polyphasic approach. This included a phylogenetic analysis based on 16S rRNA gene sequences and multi-locus sequence analysis (MLSA) based on pyrH, recA, rpoA, gyrB and mreB genes. The isolates shared 98.0 % 16S rRNA gene sequence similarity with Vibrio xiamenensis G21T. Phylogenetic analysis based on 16S rRNA gene sequences using the neighbour-joining and maximum-likelihood methods showed that the isolates fell within the genus Vibrio and formed an independent branch close to V. xiamenensis G21T. The maximum-parsimony method grouped the isolates to V. xiamenensis G21T but forming two clearly separated branches. Phylogenetic trees based on individual pyrH, recA, rpoA, gyrB and mreB gene sequences revealed that strain IGJ1.11T formed a clade alone or with V. xiamenensis G21T. Sequence similarities of the pyrH, recA, rpoA, gyrB and mreB genes between strain IGJ1.11T and V. xiamenensis G21T were 86.7, 85.7, 97.3, 87.6 and 84.8 %, respectively. MLSA of concatenated sequences showed that strain IGJ1.11T and V. xiamenensis G21T are two clearly separated species that form a clade, which we named Clade Xiamenensis, that presented 89.7 % concatenated gene sequence similarity, i.e. less than 92 %. The major cellular fatty acids (>5 %) of strain IGJ1.11T were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and summed feature 8 (C18 : 1ω7c and/or C18 : 1ω6c). Enzymic activity profiles, sugar fermentation patterns and DNA G+C content (52.9 mol%) differentiated the novel strains from the closest related members of the genus Vibrio. The name Vibrio olivae sp. nov. is proposed for the novel species. The type strain is IGJ1.11T ( = CECT 8064T = DSM 25438T).

Expression of genes involved in metabolism of phenolic compounds by Lactobacillus pentosus and its relevance for table-olive fermentations

Genes with the potential to code for enzymes involved in phenolic compound metabolism were detected in the genome of Lactobacillus pentosus IG1, isolated from a green olive fermentation. Based on homology, these genes could code for a 6-P-β Glucosidase, two different Tannases, a Gallate decarboxylase and a p-Coumaric decarboxylase. Expression of up to seven of these genes was studied in L. pentosus IG1 (olive fermentation) and CECT4023T (corn silage), including responses upon exposure to relevant phenolic compounds and different olive extracts. Genes potentially coding Tannase, Gallate decarboxylase and p-Coumaric acid decarboxylase significatively increased their expression upon exposure to such compounds and extracts, although it was strain dependent. In general, both the genetic organization and the characteristics of gene expression resembled very much those described for Lactobacillus plantarum. In accordance to the observed induced gene expression, metabolism of specific phenolic compounds was achieved by L. pentosus. Thus, methyl gallate, gallic acid and the hydroxycinamic acids p-coumaric, caffeic and ferulic were metabolized. In addition, the amount of phenolics such as tyrosol, oleuropein, rutin and verbascoside included in a minimal culture medium was noticeably reduced, again dependent on the strain considered.